CRISPR-Cas9 has demonstrated incredible potential to provide clinical benefit, but the challenge of delivery currently hinders therapeutic use of genome editing in vivo. Use of viral vectors and lipid nanoparticles has established the viability of in vivo genome editing, but these technologies have substantial drawbacks. Viral vectors are immunogenic, difficult to manufacture, and have been associated with increased risks of off-target editing. Lipid nanoparticles are unsuitable for systemic administration if targeting organs other than the liver. Ex vivo therapies relying on autologous transplantation have shown the immense value in genetic manipulation of immune cells, but the procedures remain risky, resource-intensive, and prohibitively expensive. An ideal method to deliver therapeutic genome editing enzymes would be non-toxic, compatible with intravenous administration, amenable to large-scale manufacture, and targeted to the cell type in need of genetic correction. With all this in mind, we propose delivery of CRISPR-Cas9 in the form of an RNA-protein (RNP) complex. Cas9 RNP has been shown to be safe and effective in vivo following local administration, and we have established a strategy to enable cell type-specific delivery of Cas9 RNP tethered to a molecular targeting agent (MTA) such as a receptor-binding ligand, antibody, or aptamer. Our proposal aims to use MTA-tethered Cas9 RNP for targeted editing of T cells in vivo. We will rely on established and novel MTAs to promote efficient and specific uptake of Cas9 RNP into T cells. Well- characterized antibody MTAs will direct specific editing in human, mouse, and primate T cells. Novel aptamer MTAs will be screened with a focus on cross-species reactivity to streamline the transition from pre-clinical to clinical development. Because the Cas9 RNP has no inherent ability to cross cellular membranes, it will be augmented with the ability to escape the endosome to avoid lysosomal degradation following MTA-induced endocytosis. We have established a novel modular approach to functionalize Cas9 for endosomal escape, facilitating re-optimization for specific cell types as needed. In the UG3 phase, we will complete the following three aims: (1) Enable in vivo-compatible genome editing of immune cells using targeted Cas9 RNP; (2) Identify robust molecular targeting agents for T cell-specific editing; (3) Use targeted Cas9 RNP for in vivo genome editing of T cells. Following independent validation of editing in mice, the UH3 phase will perform the following: (1) Scale up production of targeted Cas9 RNP for large animal testing; (2) Validate targeted Cas9 RNP for in vivo genome editing in non-human primates. The intersection of MTA-based cell targeting and the efficient endosomal escape of Cas9 RNP will generate a versatile genome editing platform suitable for intravenous administration. Successful completion of the proposed work will result in an engineered Cas9 RNP system that is safe, effective in vivo, readily manufactured, and ?plug & play? regarding its molecular targeting to multiple cell types of interest.